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Ceramide-rich platforms (CRPs) mediate association of proteins with the sphingolipid ceramide and may regulate protein interaction in membrane contact sites to the cytoskeleton, organelles, and infectious pathogens. However, visualization of ceramide association to proteins is one of the greatest challenges in understanding the cell biology of ceramide. Here we introduce a novel labeling technique for ceramide-associated proteins (CAPs) by combining photoactivated cross-linking of a bioorthogonal and bifunctional ceramide analog, pacFACer with proximity ligation assays (PLAs). pacFACer cross-linked to CAPs is covalently attached to a fluorophore using click chemistry. PLAs use antibodies to: (1) the candidate CAP and the fluorophore (PLA1); and (2) the CAP and ceramide (PLA2). PLA1 shows the subcellular localization of a particular CAP that is cross-linked to pacFACer, while PLA2 tests if the cross-linked CAP forms a complex with endogenous ceramide. Two proteins, tubulin and voltage-dependent anion channel 1 (VDAC1), were cross-linked to pacFACer and showed PLA signals for a complex with ceramide and pacFACer, which were predominantly colocalized with microtubules and mitochondria, respectively. Binding of tubulin and VDAC1 to ceramide was confirmed by coimmunoprecipitation assays using anti ceramide antibody. Cross-linking to pacFACer was confirmed using click chemistry-mediated attachment of biotin and streptavidin pull-down assays. Inhibition of ceramide synthases with fumonisin B1 (FB1) reduced the degree of pacFACer cross-linking and complex formation with ceramide, while it was enhanced by amyloid beta peptide (Aβ). Our results show that endogenous ceramide is critical for mediating cross-linking of CAPs to pacFACer and that a combination of cross-linking with PLAs (cross-link/PLA) is a novel tool to visualize CAPs and to understand the regulation of protein interaction with ceramide in CRPs.

Prior work suggests that amyloid precursor protein (APP) can function as a proinflammatory receptor on immune cells, such as monocytes and microglia. Therefore, we hypothesized that APP serves this function in microglia during Alzheimer9s disease. Although fibrillar amyloid β (Aβ)-stimulated cytokine secretion from both wild-type and APP knock-out (mAPP −/− ) microglial cultures, oligomeric Aβ was unable to stimulate increased secretion from mAPP −/− cells. This was consistent with an ability of oligomeric Aβ to bind APP. Similarly, intracerebroventricular infusions of oligomeric Aβ produced less microgliosis in mAPP −/− mice compared with wild-type mice. The mAPP −/− mice crossed to an APP/PS1 transgenic mouse line demonstrated reduced microgliosis and cytokine levels and improved memory compared with wild-type mice despite robust fibrillar Aβ plaque deposition. These data define a novel function for microglial APP in regulating their ability to acquire a proinflammatory phenotype during disease. SIGNIFICANCE STATEMENT A hallmark of Alzheimer9s disease (AD) brains is the accumulation of amyloid β (Aβ) peptide within plaques robustly invested with reactive microglia. This supports the notion that Aβ stimulation of microglial activation is one source of brain inflammatory changes during disease. Aβ is a cleavage product of the ubiquitously expressed amyloid precursor protein (APP) and is able to self-associate into a wide variety of differently sized and structurally distinct multimers. In this study, we demonstrate both in vitro and in vivo that nonfibrillar, oligomeric forms of Aβ are able to interact with the parent APP protein to stimulate microglial activation. This provides a mechanism by which metabolism of APP results in possible autocrine or paracrine Aβ production to drive the microgliosis associated with AD brains.

Accumulation of aggregated amyloid-β protein (Aβ) is an important feature of Alzheimer’s disease. There is significant interest in understanding the initial steps of Aβ aggregation due to the recent focus on soluble Aβ oligomers. In vitro studies of Aβ aggregation have been aided by the use of conformation-specific antibodies which recognize shape rather than sequence. One of these, OC antiserum, recognizes certain elements of fibrillar Aβ across a broad range of sizes. We have observed the presence of these fibrillar elements at very early stages of Aβ incubation. Using a dot blot assay, OC-reactivity was found in size exclusion chromatography (SEC)-purified Aβ(1-42) monomer fractions immediately after isolation (early-stage). The OC-reactivity was not initially observed in the same fractions for Aβ(1-40) or the aggregation-restricted Aβ(1-42) L34P but was detected within 1–2 weeks of incubation. Stability studies demonstrated that early-stage OC-positive Aβ(1-42) aggregates were resistant to 4M urea or guanidine hydrochloride but sensitive to 1% sodium dodecyl sulfate (SDS). Interestingly, the sensitivity to SDS diminished over time upon incubation of the SEC-purified Aβ(1-42) solution at 4° C. Within 6–8 days the OC-positive Aβ42 aggregates were resistance to SDS denaturation. The progression to, and development of, SDS resistance for Aβ(1-42) occurred prior to thioflavin T fluorescence. In contrast, Aβ(1-40) aggregates formed after 6 days of incubation were sensitive to both urea and SDS. These findings reveal information on some of the earliest events in Aβ aggregation and suggest that it may be possible to target early-stage aggregates before they develop significant stability.